Methods for labeling beta-amyloid plaques and neurofibrillary tangles

ABSTRACT

A method for labeling β-amyloid plaques and neurofibrillary tangles in vivo and in vitro, comprises contacting a compound of formula (I):  
                 
 
     with mammalian tissue. In formula (I), R 1  is selected from the group consisting of —C(O)-alkyl, —C(O)-alkylenyl-R 4 , —C(O)O-alkyl, —C(O)O-alkylenyl-R 4 , —C═C(CN) 2 -alkyl, —C═C(CN) 2 -alkylenyl-R 4 ,  
                 
 
     R 4  is a radical selected from the group consisting of alkyl, substituted alkyl, aryl and substituted aryl; R 5  is a radical selected from the group consisting of —NH 2 , —OH, —SH, —NH-alkyl, —NHR 4 , —NH-alkylenyl-R 4 , —O-alkyl, —O-alkylenyl-R 4 , —S-alkyl, and —S-alkylenyl-R 4 ; R 6 is a radical selected from the group consisting of —CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R 4 , —C(O)-alkyl, —C(O)-alkylenyl-R 4 , —C(O)-halogen, —C(O)NH 2 , —C(O)NH-alkyl, —C(O)NH-alkylenyl-R 4 ; R 7  is a radical selected from the group consisting of O, NH, and S; and R 8  is N, O or S. R 2  and R 3  are each independently selected from the group consisting of alkyl and alkylenyl-R 10 , wherein R 10  is selected from the group consisting of —OH, —OTs, halogen, spiperone, spiperone ketal and spiperone-3-yl. Alternatively, R 2  and R 3  together form a heterocyclic ring, optionally substituted with at least one radical selected from the group consisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R 10 , carbonyl, spiperone, spiperone ketal and spiperone-3-yl. In the compounds of formula (I), one or more of the hydrogen, halogen or carbon atoms can, optionally, be replaced with a radiolabel.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority of U.S. Provisional PatentApplication No. 60/097,320, filed Aug. 20, 1998, the entire disclosureof which is incorporated herein by reference.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No.DE-FC0387-ER60615, awarded by the Department of Energy. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Alzheimer's disease affects approximately 20 to 40% of thepopulation over 80 years of age, the fastest growing age group in theUnited States and other post-industrial countries. Common features inthe brain of patients with Alzheimer's disease include the presence ofabundant intraneuronal neurofibrillary tangles (NFTs) and extracellularamyloid rich β-amyloid plaques. NFTs are cytoskeletal pathologieslargely composed of aggregates of hyperphosphorylated tau proteinsassembled into periodically restricted amyloid fibers called pairedhelical filaments. The major component of amyloid plaques is a peptide,a small 39-43 aminoacid long β-amyloid peptide that is generated fromthe cleavage of a larger amyloid precursor protein. However, except fordiffuse plaques formed almost exclusively of B-amyloid peptides, amyloidplaques are complex lesions containing numerous associated cellularproducts. Mutations causing increased production of the 42 amino acidform of this peptide have been genetically linked to autosomal dominantfamilial forms of Alzheimer's diseases. Deposits of β-amyloid occur veryearly in the disease process, long before clinical symptoms develop.Because these mutations appear to be pathogenic and cause Alzheimer'sdiseases in transgenic mice, β-amyloids are widely believed to play acausal role in the disease. Whether or not amyloid deposits are causal,they are certainly a key part of the diagnosis. Further, because amyloidplaques occur early in the disease, the ability to image deposits wouldprovide a convenient marker for early diagnosis and prevention of thedisease as well as a method for monitoring the effectiveness oftherapeutic regimens.

[0004] Alzheimer's disease is currently definitively diagnosed by takingsections from postmortem brain and quantifying the density ofneocortical amyloid deposits. Unfortunately, current techniques fordetecting amyloid deposits and/or NFTs require postmortem or biopsyanalysis. For example, thioflavin fluorescent-labeling of amyloid inbrain sections in vitro is currently a widely-used method for evaluationof the brain. Another potential amyloid probe, Chrysamine-G, a congo redderivative, has also been developed. Congo red is a charged molecule andthus lacks sufficient hydrophobicity for diffusion through the bloodbrain barrier and is therefore not useful as an in vivo label. See Klunket al, Neurobiology of Aging, 16:541-548 (1995), and PCT Publication No.WO 96/34853. Chrysamine G enters the blood brain barrier better thanCongo red, but its ability to label amyloid plaques in Alzheimer's brainappears weak. See for example, H. Han, C-G Cho and P. T. Lansbury, Jr J.Am. Chem. Soc. 118, 4506 (1996); N. A. Dezutter et al, J. Label. Compd.Radiopharm. 42, 309 (1999). Similarly, earlier attempts to usemonoclonal antibodies as probes for in-vivo imaging of β-amyloid werehampered by their limited ability to cross the blood brain barrier. SeeR. E. Majocha et al, J. Nucl. Med. 33, 2184 (1992). More recently, theuse of monobiotinylated conjugates of 1251-Aβ 1-40 with permeabilitythrough the blood brain barrier has also been proposed (See Y. Saito etal., Proc. Natl. Acad. Sci. USA 22, 2288 (1991)), but its ability tolabel β-amyloid plaques and/or NFTs in vivo has not yet beendemonstrated. Quantitation of the deposits in vivo is not yet possiblewith the currently available probes. Accordingly, a need exists for aconvenient marker for early diagnosis of Alzheimer's disease.

[0005] In vivo, non invasive determination of regional cerebral glucosemetabolic rates (rCMRG1) with positron emission tomography (PET) hasbeen an important tool in the assessment of brain function inAlzheimer's disease patients. Numerous studies using2-[F-18]fluoro-2-deoxy-D-glucose (FDG) have demonstrated acharacteristic metabolic pattern of hypometabolism in temporoparietaland frontal association areas. A few of these studies have comparedrCMRGI with postmortem regional neuronal pathology. These results andthe uncertainties of the Alzheimer's disease pathogenic cascadehighlight the importance of assessing amyloid and neurofibril depositionin vivo, non-invasively in these patients.

SUMMARY OF THE INVENTION

[0006] The present invention provides methods for labeling structures,including β-amyloid plaques and neurofibrillary tangles, in vivo and invitro, and comprises contacting a compound of formula (I):

[0007] with mammalian tissue. In formula (I), R₁ is selected from thegroup consisting of —C(O)-alkyl, —C(O)-alkylenyl-R₄, —C(O)O-alkyl,—C(O)O-alkylenyl-R₄, —C═C(CN)₂-alkyl, —C═C(CN)₂-alkylenyl-R₄,

[0008] R₄ is a radical selected from the group consisting of alkyl,substituted alkyl, aryl and substituted aryl; R₅ is a radical selectedfrom the group consisting of —NH₂, —OH, —SH, —NH-alkyl, —NHR₄,—NH-alkylenyl-R₄, —O-alkyl, —O-alkylenyl-R₄, —S-alkyl, and—S-alkylenyl-R₄; R₆ is a radical selected from the group consisting of—CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)-halogen, —C(O)NH₂, —C(O)NH-alkyl,—C(O)NH-alkylenyl-R₄; R₇ is a radical selected from the group consistingof O, NH, and S; and R₈ is N, O or S.

[0009] In formula (I), R₂ and R₃ are each independently selected fromthe group consisting of alkyl and alkylenyl-R₁₀, wherein R₁₀ is selectedfrom the group consisting of —OH, —OTs, halogen, spiperone, spiperoneketal and spiperone-3-yl. Alternatively, R₂ and R₃ together form aheterocyclic ring, optionally substituted with at least one radicalselected from the group consisting of alkyl, alkoxy, OH, OTs, halogen,alkylenyl-R₁₀, carbonyl, spiperone, spiperone ketal and spiperone-3-yl.In the compounds of formula (I), one or more of the hydrogen, halogen orcarbon atoms can, optionally, be replaced with a radiolabel.

[0010] For in vitro detection of β-amyloid plaques and neurofibrillarytangles in brain tissue, the plaques are labeled, and the brain tissueis then observed with a fluorescence microscope. For in vivo detection,the β-amyloid plaques and neurofibrillary tangles in brain tissue arelabeled, preferably by injection of a solution containing a radiolabeledcompound of formula (I). The locations of the labeled β-amyloid plaquesand neurofibrillary tangles are then observed by any method capable ofdetecting and depicting the distribution of the radiolabeled compoundwithin the body.

[0011] According to the methods of the invention, amyloid deposits incryostat and paraffin sections of Alzheimer-diseased (AD) brain tissueare labeled with a level of sensitivity similar to thioflavin S. Use ofthe present invention, however, has several advantages over usingthioflavin S. Namely, no pretreatments are required. Moreover, unlikewith thioflavin S, the methods work with minimal washing and withoutformalin or paraformaldehyde fixation or differentiation of tissue.Additionally, stock solution can be kept in the freezer for six monthsand still produce acceptable results at 1/100 to 1/1,000 dilutions,eliminating the need to make the stock up fresh, as is required forthioflavin S labeling.

[0012] Systemically injected compositions according to the inventionreadily penetrate the blood brain barrier and label amyloid deposits andneurofibrillary tangles demonstrating the ability of the presentcompositions to act as an in vivo imaging probe. The methods of theinvention achieve in vivo labeling and detection of β-amyloid plaquesand neurofibrillary tangles in the brain of a living patient. Themethods of the invention not only permit detection of Alzheimer'sdisease, but also provide a way for physicians to monitor the progressof patients undergoing treatment for the disease. Thus, physicians canbetter determine whether a particular treatment method is successful andworthwhile.

[0013] In still another embodiment, the invention is directed to acomposition comprising a compound of formula (I):

[0014] R₁ is selected from the group consisting of —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C═C(CN)₂-alkyl,—C═C(CN)₂-alkylenyl-R₄,

[0015] R₄ is a radical selected from the group consisting of alkyl,substituted alkyl, aryl and substituted aryl; R₅ is a radical selectedfrom the group consisting of —NH₂, —OH, —SH, —NH-alkyl, —NHR₄,—NH-alkylenyl-R₄, —O-alkyl, —O-alkylenyl-R₄, —S-alkyl, and—S-alkylenyl-R₄; R₆ is a radical selected from the group consisting of—CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)-halogen, —C(O)NH₂, —C(O)NH-alkyl,—C(O)NH-alkylenyl-R₄; R₇ is a radical selected from the group consistingof O, NH, and S; R₈ is N, O or S; R₂ is selected from the groupconsisting of alkyl and alkylenyl-R₅ and R₃ is alkylenyl-R₅, and R₅ isselected from the group consisting of —OH, —OTs, halogen, spiperone,spiperone ketal, and spiperone-3-yl, or R₂ and R₃ together form aheterocyclic ring, optionally substituted with at least one radicalselected from the group consisting of alkyl, alkoxy, OH, OTs, halogen,alkylenyl-R₅, carbonyl, spiperone, spiperone ketal and spiperone-3-yl.One or more of the hydrogen, halogen or carbon atoms can optionally bereplaced with a radiolabel.

[0016] The invention is more preferably related to a compositioncomprising a compound of formula (II):

[0017] R₂ is selected from the group consisting of alkyl andalkylenyl-R₁₀ and R₃ is alkylenyl-R₁₀, wherein R₁₀ is selected from thegroup consisting of —OH, —OTs, halogen, spiperone, spiperone ketal andspiperone-3-yl, or R₂ and R₃ together form a heterocyclic ring,optionally substituted with at least one radical selected from the groupconsisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R₁₀, carbonyl,spiperone, spiperone ketal and spiperone-3-yl, and R₉ is an alkyl, arylor substituted aryl group, and to pharmaceutically acceptable salts andsolvates thereof. One or more of the hydrogen, halogen or carbon atomscan optionally be replaced with a radiolabel.

DESCRIPTION OF THE DRAWINGS

[0018] The file of this application contains at least one drawingexecuted in color. Copies of this patent with color drawings(s) will beprovided by the Patent an Trademark Office upon request and payment ofthe necessary fee.

[0019] These and other features and advantages of the present inventionwill be better understood by reference to the following detaileddescription when considered in conjunction with the accompanyingdrawings wherein:

[0020]FIG. 1A shows2-(1,1-dicyanopropen-2-yl)-6-dimethylaminonaphthalene (DDNP)fluorescence (ex 190 nm, em 520-530 nm) of amyloid plaques labeled inthe cortex of the brain of an Alzheimer's disease patient (X400).

[0021]FIG. 1B shows strong DDNP labeling of plaques and weak DDNPlabeling of tangles in the cortex of the brain of an Alzheimer's diseasepatient (X640).

[0022]FIG. 1C shows DDNP labeling of a single, large plaque with anamyloid core in human brain (X640).

[0023]FIG. 1D shows DDNP labeling of a plaque in agent Tg2576 HuAPPswtransgenic mouse brain (X500).

[0024]FIG. 1E shows Thioflavin S labeling of a cored plaque inAlzheimer's disease human brain (X640).

[0025]FIG. 1F shows 4G8 antibody labeling amyloid β-protein of a sliceof the same human brain shown in FIG. 1E (X640).

[0026]FIG. 2A shows labeling of amyloid injected into rat brain, wherean aliquot of β-amyloid 1-40 was allowed to aggregate for 8 days at 37°C., dried onto a gelatin coated slide, and labeled with DDNP,demonstrating fibrillar fluorescence consistent with amyloid.

[0027]FIG. 2B shows labeling of amyloid injected into rat brain, where 8days after unilateral stereotaxic injection of 3 μg of aggregatedβ-amytoid 1-40 into rat cortex, the rats were injected with 100 μL of640 μM DDNP into the carotid artery, anesthetized, and sacrificed byperfusion after 20 minutes and the brains were cryosectioned andexamined for fluoroescence; FIG. 2B demonstrates in vivo DDNPfluorescently labeled amyloid at the tip of the need track (X100).

[0028]FIG. 2C shows a high power view of the in vivo DDNP labeledmaterial of FIG. 2B (X200).

[0029]FIG. 2D depicts how formic acid treatment of a section through theinjection site removes fluorescent labeling (X100).

[0030]FIG. 2E demonstrates that DDNP labeling is weak contralateral tothe amyloid injection site, where no amyloid is present (X200).

[0031]FIG. 3A is a PET-[F-18]FDDNP(2-(1.1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene)image of a brain cross-section through thehippocampus-amygdala-entorhinal/temporal cortex region of an Alzheimer'sdisease patient.

[0032]FIG. 3B is a PET-FDG (FDG is 2-[F-18]fluoro-2-deoxy-D-glucose)image of the brain cross-section of FIG. 3A.

[0033]FIG. 3C is an MRI image (proton relaxation times) of the braincross-section of FIG. 3A.

[0034]FIG. 4 is a graph showing the estimated residence times of[F-18]FDDNP in pateints.

[0035]FIG. 5 shows an image (central image) obtained by immunostaining aforty five micrometer cryostate temporal cortex section of anAlzheimer's disease patient incubated with AT8 (anti-phosphotau) and10G4 (anti-AB1-15) at 1:800. Insets are adjacent sections of the sameAlzheimer's disease brain specimen stained with FDDNP showing, beginningin the upper left comer and moving clockwise, (1) neuritic plaques, (2)diffuse plaque, (3) vascular amyloid, (4) dense plaques and tangles, and(5) dense tangles.

DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention is directed to methods for labelingstructures such as β-amyloid plaques and neurofibrillary tangles in vivoand in vitro. The methods all involve contacting a compound of formula(I):

[0037] with mammalian tissue. In formula (I), R₁ is selected from thegroup consisting of —C(O)-alkyl, —C(O)-alkylenyl-R₄, —C(O)O-alkyl,—C(O)O-alkylenyl-R₄—C═C(CN)₂-alkyl, —C═C(CN)₂-alkylenyl-R₄

[0038] R₄ is a radical selected from the group consisting of alkyl,substituted alkyl, aryl and substituted aryl. R₅ is a radical selectedfrom the group consisting of —NH₂, —OH, —SH, —NH-alkyl, —NHR₄,—NH-alkylenyl-R₄, —O-alkyl, —O-alkylenyl-R₄, —S-alkyl, and—S-alkylenyl-R₄. R₆ is a radical selected from the group consisting of—CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)-halogen, —C(O)NH₂, —C(O)NH-alkyl,—C(O)NH-alkylenyl-R₄. R₇ is a radical selected from the group consistingof O, NH, and S. R₈ is N, O or S.

[0039] In formula (I), R₂ and R₃ are each independently selected fromthe group consisting of alkyl and alkylenyl-R₁₀, wherein R₁₀ is selectedfrom the group consisting of —OH, —OTs, halogen, spiperone, spiperoneketal and spiperone-3-yl. Alternatively, R₂ and R₃ together form aheterocyclic ring, optionally substituted with at least one radicalselected from the group consisting of alkyl, alkoxy, OH, OTs, halogen,alkylenyl-R₁₀, carbonyl, spiperone, spiperone ketal and spiperone-3-yl.In the compounds of formula (I), one or more of the hydrogen, halogen orcarbon atoms may optionally be replaced with a radiolabel.

[0040] In a preferred embodiment, the methods of the invention involvecontacting a compound of formula (II):

[0041] with mammalian tissue. In formula (II), R₂ and R₃ are as definedabove, and R₉ is an alkyl, aryl or substituted aryl group.

[0042] As used herein, the term “alkyl” refers to a straight or branchedchain monovalent radical of saturated carbon atoms and hydrogen atoms,such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,pentyl, and hexyl. The term “alkylenyl” refers to a divalent analog ofan alkyl group, i.e., methylenyl (—CH₂—), ethylenyl (—CH₂CH₂—), etc. Theterm “aryl” refers to a mono- or polycyclic substituted or unsubstitutedaromatic ring.

[0043] As used herein, the term “lower alkyl” refers to a straight orbranched chain monovalent radical having from one to four saturatedcarbon atoms and hydrogen atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and t-butyl.

[0044] As used herein, the term “heterocyclic ring” refers to anon-aromatic, monocyclic or bicyclic radical containing 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 ring atoms, each of which is saturated orunsaturated, including 1, 2, 3, 4, or 5 heteroatoms selected fromnitrogen, oxygen and sulfur. Nonlimiting examples include aziridine,azetidine, pyrrolidine, piperidine, piperizine and derivatives thereof.Preferably, these heterocyclic rings are substituted with alkyl groupsor substituted alkyl groups, for example, alkyl groups havingsubstituents such as those defined for R₄ above.

[0045] For the compounds of formula (I) and formula (II), preferably R₂and R₃ are each independently alkyl, more preferably lower alkyl. Forthe compounds of formula (II), preferably R₉ is lower alkyl, morepreferably methyl or ethyl, aryl and substituted aryl. Particularlypreferred compounds for use in connection with the invention are2-(1,1-dicyanopropen-2-yl)-6-dimethylaminonaphthalene (DDNP) and2-(1,1-dicyanopropen-2-yl)-6-(ethyl)(methyl)(amino)-naphthalene, both ofwhich can be optionally radiolabeled. Another preferred compound,particularly for use in vivo, is2-(1,1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene([F-18]FDDNP).

[0046] The present invention is also directed to methods for detectingstructures, such as β-amyloid plaques and neurofibrillary tangles invitro and in vivo. The term “structures” refers to aggregates ofbiological materials containing peptides and other cellular materialsthat may occur as part of a disease pathology. The term “peptides”includes proteins.

[0047] The compounds described above have fluorescent activity in therange of about 470 to 610 nm. In one application, the present inventionlabels β-amyloid plaques and neurofibrillary tangles in brain tissue.Accordingly, for in vitro detection Alzheimer's disease, the compoundsare contacted with brain tissue, and the brain tissue observed with afluorescence microscope.

[0048] For in vivo detection, preferably the compounds are radiolabeled.A preferred radiolabel is ¹⁸F, which has a half-life of approximatelytwo hours for position emission tomography (PET). Another radiolabel isradioiodine, for example, ¹²³I for use with single photon emissioncomputed tomography (SPECT). Alternatively, other radiolabels are used,such as ¹¹C, ¹³N and ¹⁵O, although these radiolabels are less desirabledue to their relatively short half-lives. Any atom in the compound canbe replaced with a suitable radiolabel. Radiolabeling can be achieved byany method known to those skilled in the art. For example, dry[F-18]fluoride ion [¹⁸O(p,n)¹⁸F] in K₂CO₃ (0.75 mg) and Kryptofix 2.2.2™(19 mg) are added to a solution of the compound of formula (I) orformula (II) (4 mg in 1 mL CH₃CN). The mixture is heated in an oil bathat 85° C. for about 10 to 40 minutes. After cooling and dilution withwater, the radiolabeled product can be purified by preparative HPLC.Kryptofix 2.2.2™ is a crown ether, available from Aldrich Chemical Co.(Milwaukee, Wis.).

[0049] A solution containing the radiolabeled compound is then injectedinto the patient. As used herein, the term “patient” refers to anymammal, including humans, rats, mice, dogs and cats. Neuroanatomicalregions can be determined manually using MRI scans, for example, using aTela magnet, and then on amyloid-PET (positron emission tomography) andFDG-PET (fluorodeoxyglucose-PET) by coregistration of the MRI scans. PEThas current resolution of 2 to 3 min, a dynamic determination ofradiolabeled compound deposition in the brain, and permits detection ofabnormal areas.

[0050] By the above-described methods, diseases characterized by theaccumulation of β-amyloid plaques and neurofibrillary tangles such asAlzheimer's disease and other diseases associated with braindeterioration, can be detected.

EXAMPLES Example 1

[0051] The following compositions according to the invention wereprepared. NMR spectra were obtained on Bruker AM 360 WB or DPX 300Spectrometers. ¹H chemical shifts are reported in ppm downfield from TMSas an internal standard. ¹⁹F chemical shifts are reported relative toexternal fluorotrichloromethane. Deuteriochloroform was used as thesolvent unless stated otherwise. Melting points were determined on anElectrothermal Melting Point Apparatus and are uncorrected. Elementalanalyses were performed by Galbraith Laboratories, Inc., Knoxville,Tenn. or Ms. Metka Kastelic at the Faculty of Chemistry and ChemicalTechnology, University of Ljubljana. Radial chromatography was performedon Chromatotron (Harrison Research, 840 Moana Court, Palo Alto, Calif.94306). The rotors were prepared as recommended by Harrison Researchusing E. Merck Silica Gel (Cat. No. 7749-3). HPLC was performed on anAlltech Econosil C-18 5 μm, 4.6×250 mm column using a 40:60:2 mix ofwater:acetonitrile:triethyl amine as the solvent UV detection at 254 nmwas used. Solvents and reagents were from Fisher, Aldrich or Fluka andwere used as received unless noted otherwise.

Example 1(a) Preparation of2-(1,1-dioyanopropen-2-yl)-6-dimethylaminonaphthalene (DDNP)

[0052]

[0053] To a solution of 5.26 g (117 mmol) of dimethylamine in 29 mL offreshly distilled hexamethylphosphoric triamide (HMPT) were added 31 mLof dry toluene and 780 mg (112 mmol) of Li in small pieces. The mixturewas stirred under argon at room temperature for 1.5 hours.2-Acetyl-6-methoxynaphthalene was prepared as described in Arsenijevicet al., Org. Synth. Coll. 1988, 6:34-36, the disclosure of which isincorporated herein by reference. 2-Acetyl-6-methoxynaphthalene (5.57 g,27.8 mmol) was added in one portion, and stirring was continued for 20hours. The mixture was cooled in an ice-water bath and poured into acold water/ethyl acetate mixture (300 mL each). After thorough mixing,the layers were separated, and the water layer was extracted twice with225 mL of ethyl acetate. Organic extracts were combined, dried, andevaporated to give a yellow solid. Recrystallization from ethanolafforded 3.67 g (64%) of 2-acetyl-6-(dimethylamino)naphthalene (ADMAN)as a yellow solid, melting at 153.5-155° C.: ¹H NMR (CDCl₃, TMS) δ2.67(s, 3H, COCH₃), 3.15 (s, 6H, N(CH₃)₂), 6.87 (d, 1H, H-5), 7.17 (dd, 1H,H-7), 7.63 (d, 1H, H-4),7.80 (d, 1H, H-8), 7.92 (dd, 1H, H-3), 8.32 (bs,1H, H-1). J_(1,3)=2.3 Hz, J_(3,4)=8.7 Hz, J_(5,7)=2.4 Hz, J_(7,8)=9.3Hz. MS (M⁺) 213: found: 213. Anal. Calcd for C₁₄H₁₅NO: C, 78.84; H,7.09; N, 6.57. Found C, 78.96; H, 7.10; N, 6.45.

[0054] A mixture of malonitrile (436 mg, 6.6 mmol) and ADMAN (1.278 g,6.6 mmol) was heated to 110° C. in 20 mL of pyridine for 19 hours. Aftercooling, the remaining red solid was dissolved in 100 mL of methylenechloride, adsorbed onto 10 g of flash silica get (230-400 mesh) andchromatographed with toluene. Appropriate fractions were combined andevaporated to give 1.12 g (72%) of2-(1,1-dicyanopropen-2-yl)-6-dimethylaminonaphthalene (DDNP).Recrystallization from benzene-hexane gave red needles melting at154.5-155° C.: ¹H NMR (CDCl₃, TMS) δ2.69 (s, 3H, CH₃), 3.11 (s, 6H,N(CH₃)₂), 6.85 (d, 1H, H-5), 7.18 (dd, 1H, H-7), 7.56 (dd, 1H, H-3),7.66 (d, 1H, H-4), 7.76 (d, 1H, H-8), 8.02 (d, 1H, H-1). J_(1,3)=2.04Hz, J_(3,4)=9.13 Hz, J_(5,7)=2.5 Hz, J_(7,8)=9.11 Hz. IR (CHCl₃) 2250cm⁻¹ (CN stretching). MS (M⁺) 261: found: 262. Anal. Calcd for C₁₇H₁₅N₃:C, 78.13; H, 5.79; N, 18.08. Found C, 78.17; H, 5.68; N, 17.91.

Example 1(b)

[0055] Preparation of2-(1-{6[ethyl-(2-{8-[4-(4-fluorophenyl)-4-oxobutyl]-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]dec-3-yl}ethyl)-amino]-2-naphthyl}ethylidene)malononitrile

[0056] In a 3 L two-neck round bottom flask, equipped with a refluxcondenser and a dropping funnel, 2 L of hydrochloric acid (d=1.16) werestirred and heated to boiling. A solution of 6.06 g (30.3 mmol) of1-(6-methoxy-2-naphthyl)-1-ethanone (prepared as described inArsenijevic et al., Org. Synth. Coll. 6:34 (1988), the disclosure ofwhich is incorporated herein by reference) in a minimum amount ofdichloromethane was added, and the mixture was stirred and heated atreflux for 2 hours. The hot solution was filtered through a mineral woolplug to remove oily residue. The solid that separated after cooling wasfiltered on a glass frit and dissolved in 130 mL of ethyl acetate. Thesolution was washed with brine, dried with anhydrous magnesium sulfateand evaporated to give 5 g (89%) of 1-(6-hydroxy-2-naphthyl)1-ethanone.

[0057] A mixture of 1-(6-hydroxy-2-naphthyl)ethanone (744 mg, 3.92mmol), sodium hydrogen sulfate(IV) (1.66 g, 16 mmol),2-ethylaminoethanol (2 mL) and water (5 mL) was heated in a steel bombat 130-140° C. for 3 days. After cooling, the mixture was distributedbetween water and ethyl acetate, and the organic layer was washed withbrine, dried and evaporated. The residue was dissolved in acetone andloaded onto a 4 mm dry silica plate for radial chromatography. The platewas eluted with a 1:1 mixture of petroleum ether and ethyl acetate.Appropriate fractions were collected and evaporated to give 125mg(12%)of 1-{6-[ethyl-(2-hydroxylethyl)-amino]-2-naphthyl}ethanone.

[0058] A solution of1-{6-[ethyl-(2-hydroxylethyl)-amino]-2-naphthyl}ethanone (125 mg, 0.486mmol) in pyridine (3.5 mL) was cooled to −15° C. and p-toluenesulfonicanhydride (252 mg, 0.81 mmol) was added with stirring under argon. Thereaction mixture was allowed to slowly warm up to the room temperature,and stirring was continued for 24 hours. Because TLC (silica, 10% ethylacetate in petroleum ether) revealed that the starting material wasstill present, more p-toluenesulphonic anhydride (252 mg, 0.81 mmol) wasadded, and stirring was continued for an additional 24 hours. Themixture was then cooled in an ice-water bath and distributed betweenbrine and ether. The organic layer was dried and evaporated to leave anoily residue. The product,6-acetyl-2-(ethyl-2-[(4-methylphenyl)-sulfonyloxy]-ethylamino)-naphthalene,was isolated by radial chromatography (1 mm silica, dichloromethane) in30% yield. HRMS calcd. for C₂₃H₂₅NO₄S:411.1504. Found: 411.1514. ¹H NMRδ1.25 (t, 3H, CH₂ CH ₃), 2.33 (s, 3H, Ph-CH ₃), 2.67 (s, 3H, COCH₃),3.49 (q, 2H, CH ₂CH3), 3.75 (t, 2H, NCH ₂O), 4.25 (t, 2H, NCH₂ CH ₂O),6.97 (d, 1H, 5-H), 7.01 (dd, 1H, 7-H), 7.18 and 7.20 (d, 2H, 3′-H,5′-H), 7.56 (d, 1H, 4-H), 7.69 and 7.72 (d, 2H, 2′-H, 6′-H), 7.75 (d,1H, 8-H), 7.93 (dd, 1H, 3-H), 8.29 (d, 1H, 1-H). J_(1,3)=1.6 Hz,J_(2′,6)=J_(3′,5)=8.5 Hz, J_(7,5)=2.5 Hz, J_(7,8)=9.2 Hz, J_(3,4)=8.7Hz, J_((CH2CH3))=7.1 Hz, J_((NCH2CH2O))=6.2 Hz.

[0059] To a solution of sodium hydroxide (1 g) and tetra-n-butylammoniumhydrogensulfate (VI) (50 mg, 0.15 mmol) in water (2 mL), spiperone ketal(8-3[2-(4-fluorophenyl)-1,3-dioxolan-2yl]propyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one(which can be prepared as described in U.S. Pat. No. 3,839,342, ChemAbstr. 82:43416, and Kiesewetter et al., Appl. Radiat. Isot. 37:1181(1986), the disclosures of which are incorporated herein by reference)(15 mg, 0.034 mmol) was added and vigorously stirred. After 10 minutes,a solution of6-acetyl-2-(ethyl-2-[(4-methylphenyl)-sulfonyloxy-ethylamino)-naphthalene(12 mg, 0.03 mmol) in toluene (3 mL) was added, and the reaction mixturewas stirred and heated at 90° C. for 1 hour. After cooling, the reactionmixture was distributed between water and dichloromethane, and theorganic layer was washed with brine, dried and evaporated to leave anoily residue. Radial chromatography (1 mm silica, 2% methanol indichloromethane) yielded 5 mg (25%) of1-[6-(ethyl-2-[(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]-propyl-4-phenyl-2,4,8-triazaspiro[4.5]dec-1-en-1-yl)-oxy]-ethylamino)-2-naphthyl]1-ethanone(compound A) and 11 mg (56%) of1-[6-(ethyl-[2-(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2yl]-propyl-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]dec-2-yl)-ethyl]amino-2-naphthyl)-1-ethanone(compound B).

[0060] Compound A—HRMS calcd. for C₄₁H₄₇FN₄O₄: 678.3581. Found:678.3605. ¹H NMR: δ1.45-2.24 (m, 11H, spiperone CH₂, CH₂ CH ₃),2.35-2.84 (m, 6H, spiperone), 2.65 (s, 3H, OCH₃), 3.59 (q, 2H, NCH₂CH₃), 2.35-2.84 (M, 6H, spiperone), 2.65 (s, 3H, OCH₃), 3.59 (q, 2H,NCH ₂CH₃), 3.76 in 4.05 (m, 4H, OCH₂CH₂O), 3.85 (t, 2H, NCH ₂CH₂O), 4.52(t, 2H, OCH ₂CH₂N), 4.99 (s, 2H, NCH₂N), 6.76-6.83 9 m, 3H, phenyl,fluorophenyl), 6.93 (d, 1H, 5-H), 6.95-7.04 (M, 2H, phenyl,fluorophenyl), 7.19 (dd, 1H, 7-H), 7.21-7.26 (m, 2H, phenyl,fluorophenyl), 7.39-7.45 (m, 2H, phenyl, fluoropheynl), 7.61 (d, 1H,4-H), 7.78 (d, 1H, 8-H), 7.93 (dd, 1H, 3-H), 8.30 (d, 1H, 1-H).J_(1,3)=1.5 Hz, J_(5,7)=2.4 Hz, J_(3,4)=9.5 Hz, J_(7,8)=9.2 Hz,J_((CH2CH3))=7.1 Hz, J_((NCH2CH2O))=6.3 Hz.

[0061] Compound B—HRMS calcd. for C₄₁H₄₇FN₄O₄: 678.3581. Found:678.3603. ¹H NMR: δ1.20-1.94 (m, 17H, spiperone CH₂, CH₂ CH ₃), 2.66 (s,3H, COCH₃), 3,56 (q, 2H, NCH ₂CH₃), 3.66 and 4.02 (m, 4H, OCH₂CH₂O),3.71 3.81 (m, 4H, NCH₂CH₂N), 4.68 (s, 2H, NCH₂N), 6.82-6.90 (m, 2H,phenyl, fluorophenyl), 6.94 (d, 1H, 5-H), 6.98-7.04 (m, 2H, phenyl,fluorphenyl), 7.18 (dd, 1H, H-7), 7.21-7.26 (m, 3H, phenyl,fluorophenyl), 7.39-7.45 (m, 2H, phenyl, fluorophenyl), 7.60 (d, 1H,4-H), 7.78 (d, 1H, 8-H), 7.93 (dd, 1H, 3-H), 8.29 (d, 1H, 1-H).J_(1,3)=1.6 Hz, J_(3,4)=9.8 Hz, J_(5,7)=2.4 Hz, J_(7,8)=10.4 Hz,J_((CH2CH3))=7.1 Hz.

[0062] A solution of1-[6-(ethyl-[2-(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2yl]-propyl-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]dec-2-yl)-ethyl]amino-2-naphthyl)-1-ethanone(13 mg, 0.018 mmol) and malononitrile (6 mg, 0.09 mmol) in pyridine (3mL) was heated at 85° C. under argon for 24 hours. After pyridine wasremoved in vacuo at room temperature, the residue was distributedbetween brine and dichloromethane, and the organic layer was dried andevaporated. The product, 2-[1-(6-ethyl-[2(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]-propyl-1-oxo-4-phenyl-2,4,8-triazoaspiro[4.5]dec-2-yl)-ethyl]-amino-2-naphthyl)-ethylidene]-malononitrilewas isolated by radial chromatography (1 mm silica, 2.5% methanol indichloromethane; 13.5 mg, 97%).

[0063] HRMS calcd. for C₄₄H₄₈FN₆O₃ (M+H): 727.37719. Found: 727.3772. ¹HNMR: δ1.25-1.93 (m, 17H, spiperone CH₂, CH₂ CH ₃), 2.70 (s, 3H,C—C—CH₃), 3.57 (q, 2H, NCH₂CH₃), 3.64 and 4.03 (m, 4H, OCH₂CH₂O),3.71-3.78 (m, 4H, NCH₂CH₂N), 4.68 (s, 2H, NCH₂N), 6.83-6.88 (m, 2H,phenyl, fluorophenyl), 6.94 (d, 1H, 5-H), 6.96-7.04 (m, 2H, phenyl,fluorophenyl), 7.18 (dd, 1H, H-7), 7.21-7.25 (m, 3H, phenyl,fluorophenyl), 7.39-7.45 (m, 2H, phenyl, fluorophenyl), 7.56 (dd, 1H,3-H), 7.63 (d, 1H, 4-H), 7.76 (dd, 1H, 9-H), 8.00 (d, 1H, 1-H).J_(1,3)=1.9 Hz, J_(3,4)=8.8 Hz, J_(5,7)=2.4 Hz, J_(7,8)=9.3 Hz,J_((CH2CH3))=7.1 Hz.

[0064] The ketal protective group was removed by stirring2-[1-(6-ethyl-[2-(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]-propyl-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]dec-2-yl)-ethyl]-amino-2-naphthyl)-ethylidene]-malononitrile(13.5 mg, 0.0186 mmol) in methanol (1 mL) with one drop of concentratedhydrochloric acid for 3 hours at room temperature. The reaction mixturewas diluted with dichloromethane and washed with a saturated solution ofsodium bicarbonate. After evaporation in vacuo, the residue was purifiedby radial chromatography (1 mm silica, 2% methanol in dichloromethane)to give 10 mg (79%) of2-(1-6[ethyl-(2-8-[4-(4-fluoropheny)-4-oxobutyl]-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]dec-2-ylethyl)-amino]-2-naphthylethylidene)-malonitrile.FAB MS calcd for C₄₂H₄₄FN₆O₂ (M+H): 683.35. Found 683. ¹H NMR:δ1.21-3.02 (m,17H, spiperone CH₂, CH₃), 2.71 (s, 3H, C═C—CH₃), 3.56 (q,2H, NCH ₂CH₃) 3.9 (m, 4H, NCH₂CH₂N), 4.67 (s, 2H, NCH₂N), 6.79-7.23 (m,7H, phenyl, fluorophenyl), 6.95 (d, 1H, 5-H), 5,19 (dd, 1H, 7-H), 7.56(dd, 1H, 3-H), 7.65 (d, 1H, 4-H), 7.76 (d, 1H, 8-H), 7.97-8.04 (m, 3H,fluorophenyl, 1-H). J_(1,3)=1.9 Hz, J_(3,4)=8.8 Hz, J_(5,7)=2.5 Hz,J_(7,8)=9.1 Hz, J_((CH2CH3))=7.1 Hz.

Example 1(c)2-(1-6-[4-(8-[4-(4-Fluorophenyl)-4-oxobutyl]-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]-dec-2-ylmethyl)-piperidino]-2-naphthylethylidene)-malononitrile

[0065]

[0066] A mixture of 1-(6-hydroxy-2-naphthyl)-1-ethanone (653 mg, 3.5mmol) (prepared as described in Example 1(b)), sodiumhydrogensulfate(IV) (1.6 g, 15.5 mmol), 4-piperidylmethanol (2 g, 17.6mmol) (prepared as described in Bradbury et al., J. Med. Chem. 34:1073(1991), the disclosure of which is incorporated herein by reference),and water (6 mL) was heated in a steel bomb at 135-142° C. for 16 days.After cooling, the reaction mixture was extracted with ethyl acetate.Some product still remained in the residue, so it was further extractedwith 5% methanol in dichloromethane. Organic extracts were combined,dried and evaporated. The residue was chromatographed by radialchromatography (2 mm silica, 2% methanol in dichloromethane) to yield139 mg (14%) of1-6-[(4-hydroxymethyl)-piperidino]-2-naphthyl-1-ethanone. Afterrecrystallization from ethyl acetate the compound melted at 180-182° C.¹H NMR: δ1.44 (dddd, 2H, 3′ a-H, 5′a-H), 1.76 (m, 1H, 4′a-H), 1.91 (bd,2H, 3′e-H, 5′c-H), 2.68 (s, 3H, COCH₃), 2.89 (ddd, 2H, 2′a-H, 6′a-H),3.58 (d, 2H, OCH₂), 3.94 (bd, 2H, 2′e-H, 6′e-H), 7.10 (d, 1H, 5-H), 7.32(dd, 1H, 7-H), 7.66 (d, 1H, 4-H), 7.80 (d, 1H, 8-H), 7.94 (dd, 1H, 3-H),8.32 (d, 1H, 1-H) J_(3′a,3′e)=J_(5′a,5′e)=12.5 Hz,J_(2′a,3′a)=J_(6′a,5′a)=12.5 Hz, J_(3′a,4′a)=J_(5′a,4′a)=12.5 Hz,J_(2′e,3′a)=J_(6′a,5′a)=4.0 Hz, J_(2′a,2′c)=J_(6′a,6′e)=12.5 Hz,J_(2′a,3′e)=J_(6′a,5e)=2.6 Hz, J_(4a,OCH2)=6.4 Hz, J_(1,3)=1.9 Hz,J_(3,4)=8.9 Hz, J_(5,7)=2.3 Hz, J_(7,8)=9.0 Hz.

[0067] A solution of1-6-[(4-hydroxymethyl)-piperidino]-2-naphthyl-1-ethanone (59 mg, 0.2mmol) in pyridine (3 mL) was cooled to −15° C., and p-toluenesulfonicanhydride (205 mg, 0.6 mmol) was added with stirring under argon. Thereaction mixture was allowed to slowly warm up to the room temperatureduring 1 hour. It was cooled again and distributed between brine andether. The organic layer was washed with brine, dried and evaporated toleave 83 mg (91%) of raw1-(6-acetyl-2-naphthyl)-4-[(4-methylphenyl)-sulfonyloxy]-methylpiperidine.

[0068] To a solution of sodium hydroxide (1 g) and tetra-n-butylammoniumhydrogen-sulfate(VI) (50 mg, 0.15 mmol) in water (2 mL), spiperone ketal(100 mg, 0.2 mmol) was added and vigorously stirred. After 10 minutes, asolution of1-(6-acetyl-2-naphthyl)-4-[(4-methylphenyl)-sulfonyloxy]-methylpiperidine(98 mg, 0.2 mmol) in toluene (10 mL) was added, and the reaction mixturewas stirred at room temperature for 11 days. The reaction mixture wasdistributed between brine and dichloromethane, and the organic layer wasdried and evaporated to leave 190 mg of an oily residue. Radialchromatography (1 mm silica, dichloromethane followed by 2% methanol indichloromethane) yielded 27 mg (17%) of1-[6-(4-[(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]-propyl-4-phenyl-2,4,8-triazaspiro[4.5]dec-1-en-1-yl)-oxyl-methylpiperidino)-2-naphthyl]-1-ethanone(compound 3) and 92 mg (58%) of1-(6-4-[(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2y-l]-propyl-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]dec-2-yl)-methyl]-piperidino-2-naphthyl)-1-ethanone(compound 4).

[0069] Compound 3: HRMS calcd. for C₄₃H₄₉FN4O₄: 704.3738. Found:704.3760. ¹H NMR δ1.46-1.90 (m, 10H, 3′a-H, 5′a-H, 3′e-H, 5e-H,spiperone), 1.88 (m, 1H, 4′a-H), 2.15 and 2.38 (b, 4H, spiperone), 2.67(s, 3H, COCH₃), 2.80 (m, 4H, spiperone), 2.95 (m, 2H, 2′a-H, 6′a-H),3.75 (m, 2H, OCH₂CH₂O), 3.87 (m, 2H, 2′e-H, 6′e-H), 3.92 (m, 2H,OCH₂CH₂O), 4.19 (d, 2H, OCH₂), 4.97 (s, 2H, NCH₂N), 6.7-6.9 (m, 3H, Ph),7.01 (m, 2H, Ph), 7.11 (d, 1H, 5-H), 7.23 (m, 211, Ph), 7.32 (dd, 1H,7-H), 7.41 (m, 2H, Ph), 7.66 (d, 1H, 4-H), 7.81 (d, 1H, 8-H), 7.95 (dd,1H, 3-H), 8.32 (d 1H, 1-H), J_(2′a,2′e)=J_(6′a,6′e)=12.4 Hz,J_(2′a,3′e)=J_(6′a,5′e)=2.6 Hz, J_(4′a,OCH2)=6.1 Hz, J_(1,3)=0.1 Hz,J_(3,4)=8.8 Hz, J_(5,7)=2.1 Hz, J_(7,8)=9.1 Hz.

[0070] Compound 4: HRMS calcd. for C₄₃H₄₉FN₄O₄:704.3738. Found:704.3710. ¹H NMR δ1.50 (dddd, 2H, 3′a-H, 5′a-H), 1.55-1.70 (m, 4H,spiperone), 1.84 (bd, 2H, 3′e-H, 5′e-H), 1.92 (m, 2H, spiperone), 1.98(m, 1H, 4′a-H), 2.42 (m, 2H, spiperone), 2.67 (s, 3H, COCH₁), 2.69 (m,2H, spiperone), 2.83 (m, 4H, spiperone), 2.88 (m, 2H, 2′a-H, 6′a-H),3.35 (d, 2H, 4′-CH₂N), 3.75 (m, 2H, OCH₂CH₂O), 3.92 (bd, 2H, 2′e-H,6′e-H), 4.02 (m, 2H, OCH₂CH₂O), 4.71 (s, 2H, NCH₂N), 6.88 (m, 1H, Ph),6.91 (m, 2H, Ph), 7.01 (m, 2H, Ph), 7.08 (bs, 1H, 5-H), 7.27 (m, 3H,7-H, Ph), 7.43 (m, 2H, Ph), 7.65 (d, 1H, 4-H), 7.79 (d, 1H, 8-H), 7.94(dd, 1H, 3-H), 8.32 (bs, 1H, 1-H), J_(3′a,3′e)=J_(5′a,5′e)=12.4 Hz,J_(2′a,3′a)=12.5 Hz, J_(2′a,2e)=J_(6′a,6′e)=12.8 Hz,J_(2′a,3′e)=J_(6′a,5′e)=2.4 Hz, J_(4′a,4′)-CH₂N=7.3 Hz, J_(1,3)=1.9 Hz,J_(3,4)=8.8 Hz, J_(5,7)=2.1 Hz, J_(7,8)=9.2 Hz.

[0071] Using the procedure described in Example 1(b) for the synthesisof2-[1-(6-ethyl-[2-(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]-propyl-1-oxo-4-phenyl-2,4,8-triazoaspiro[4.5]dec-2-yl)-ethyl]-amino-2-naphthyl)-ethylidene]-malonitrile,1-(6-4-[(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2y-]-propyl-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]dec-2-yl)-methyl]-piperidino-2-naphthyl)-1-ethanonewas transformed into2-[1-(6-4-[(8-3-[2-(4-fluorophenyl)-1,3-dioxolan-2y-l]-propyl-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]dec-2-yl)-methyl]-piperidino-2-naphthyl)-ethylidene]malonitrile.It was purified by radial chromatography on a 1 mm silica plate using 2%MeOH in CH₂Cl₂ as the solvent. FAB HRMS calcd. for C₄₆H₅₀FN₆O₃ (M+H):753.3928. Found: 753.3940. ¹H NMR δ1.60-2.1 (m, 11H, spiperone, 3′a-H,3′e-H, 4′a-H, 5′a-H, 5′e-H), 2.40 (m, 2H, spiperone), 2.71 (s, 3H,C═CCH₃), 2.60-2.80 (m, 6H, spiperone), 2.91 (m, 2H, 2′a-H, 6′a-H), 3.37(d, 2H, 4′-CH₂N), 3.75 (m, 2H, OCH₂CH₂O), 3.94 (bd, 2H, 2′e-H, 6′e-H),4.02 (m, 2H, OCH₂CH₂O), 4.72 (s, 2H, NCH₂N), 6.85-6.95 (m, 3H, Ph), 7.01(m, 2H, fluorophenyl), 7.07 (d, 1H, 5-H), 7.31 (m, 3H, 7-H, Ph), 7.41(m, 2H, fluorophenyl), 7.56 (dd, 1H, 3-H), 7.69 (d, 1H, 4-H), 7.77 (d,1H, 8-H), 8.01 (d, 1H, 1H), J_(2′a,3′a)=J_(5′a,6′a)=12.8 Hz,J_(2′a,2′e)=J_(6′a,6′e)=12.8 Hz, J_(4′a,4′-CH2N)=7.6 Hz, J_(1,3)=1.8 Hz,J_(3,4)=8.6 Hz, J_(5,7)=2.2 Hz, J_(7,8)=9.4 Hz,J_(2′a,3′e)=J_(5′e,6′a)=1.8 Hz, J_(H,F)=8.7 and 6.2 Hz.

[0072] The ketal protective group was removed, as described in Example1(b), to give2-(1-6-[4-(8-[4-(4-fluorophenyl)-4-oxobutyl]-1-oxo-4-phenyl-2,4,8-triazaspiro[4.5]-dec-2-ylmethyl)-piperidino]-2-naphthylethylidene)-malononitrilein a quantitative yield. FAB HRMS calcd. for C₄₄H₄₆FN₆O₂ (M+H):709.3666. Found: 709.3689. ¹H NMR δ1.60-2.1 (m, 11H, spiperone, 3′a-H,3′e-H, 4′a-H, 5′a-H, 5′e-H), 2.5-2.71 (m, 4H, spiperone), 2.73 (s, 3H,C═C—CH₃), 2.8-3.1 (m, 6H, spiperone, 2′a-H, 6′a-H), 3.38 (d, 2H,4′-CH₂N), 3.96 (bd, 2H, 2′e-H, 6′e-H), 4.74 (s,2H, NCH₂N), 6.91 (m, 3H,phenyl), 7.1 (d, 1H, 5-H), 7.15 (m, 2H, fluorophenyl), 7.24-7.30 (m,2H,Ph), 7.34 (dd, 1H, 7-H), 7.58 (dd, 1H, 3-H) 7.72 (d, 1H, 4-H), 7.79 (d,1H, 8-H), 8.01-8.08 (m, 3H, fluorophenyl, 1-H). J_(1,3)=2.0 Hz,J_(3,4)=8.6 Hz, J_(5,7)=2.4 Hz, J_(7,8)=9.2 Hz, J_(4′a,4′-CH2N)=7.4 Hz,J_(2′a,2′e)=J_(6′a,6′e)=13.0 Hz, J_(2′a,3′a)=J_(5′a,6′a)=12.5 Hz,J_(2′a,3′e)=J_(5′e,6′a)=1.9 Hz, J_(H,F)=8.7 and 6.2 Hz.

Example 1(d) Preparation oftert-Butyl-4-(6-acetyl-2-naphthyl)-1-piperazinecarboxylate

[0073]

[0074] Anhydrous piperazine (7 g, 81.3 mmol; dried in a vacuumdesiccator over KOH-drierite mixture for 3 days) was dissolved in amixture of dry, freshly distilled toluene and hexamethyl phosphoricamide (HMPA), 25 mL each. To the solution was added 556 mg (80.1 mmol)of lithium rod, cut in small pieces under argon atmosphere, and themixture was stirred under argon for 24 hours, during which time alllithium has dissolved. Vacuum-dried 1-(6-methoxy-2-naphthyl)-1-ethanone(prepared as described in Arsenijevic et al., Org. Synth. Coll. 19886:34-36, the disclosure of which is incorporated herein by reference)(3.5 g, 17.5 mmol) was added, and stirring was continued for additional65 hours. After quenching with 300 mL of water, extraction withdichloromethane (3×300 mL), drying with anhydrous magnesium sulfate, andevaporation, a mixture of white and yellow solids was obtained.Extraction with 300 mL of hot methanol gave raw product,1-(6-piperazino-2-naphthyl)-1-ethanone, which was purified by columnchromatography (70-230 mesh silica, 25×120 mm, 5% methanol indichloromethane). The yield was 1.54 g (35%). After recrystallizationfrom ethyl acetate, the sample melted at 170.5-172° C.

[0075] 1-(6-piperazino-2-naphthyl)-1-ethanone was also prepared byheating 1-(6-hydroxy-2-naphthyl)-1-ethanone (prepared as described inExample 1(b)) (441 mg, 2.36 mmol) at 140-150° C. with 6 g piperazinehydrate (30.9 mmol) and 244 mg (2.35 mmol) NaHSO₃ for 24 hours.Additional sodium bisulfite (2 g, 19.2 mmol) was added. After anadditional 24 hours, more bisulfite (1 g) was added, and heating wascontinued (total reaction time 72 hours). After cooling, the mixture wasextracted with 2×50 mL methanol. The residue after evaporation ofmethanol was suspended in 50 mL water and extracted with ethyl acetate(5×80 mL). Combined extracts were dried (magnesium sulfate) andevaporated to give 430 mg of yellow solid. Radial chromatography (4 mmsilica, methanol) gave 83 mg (19%) of starting naphthol and 276 mg (46%;56%, based on unrecovered starting material) of1-(6-piperazino-2-naphthyl)-1-ethanone. The1-(6-piperazino-2-naphthyl)-1-ethanone was in all respects identicalwith the compound obtained using the alternative method described above.Anal. calculated for C₁₆H₁₈N₂O: C, 75.56; H, 7.13; N, 11.01. Found: C,75.82; H, 7.27; N, 10.92. ¹H NMR: δ2.68 (s, 3H, CH₃), 3.09 and 3.35 (t,J=4.95 Hz, 8H, piperazine), 7.10 (d, 1H, 5-H), 7.31 (dd, 1H, 7-H), 7.69(d, 1H, 4-H), 7.83 (d, 1H, 8-H), 7.95 (d, 1H, 3-H), 8.34 (s, 1H, 1-H);J_(5,7)=2 Hz, J_(7,8)=8.4 Hz, J_(1,3)=2 Hz, J_(3,4)=8.4 Hz.

[0076] 1-(6-piperazino-2-naphthyl)-1-ethanone (254 mg, 1 mmol) was addedto a stirred mixture of 1 g NaOH, 100 mg tetra-n-butylammoniumhydrogensulfate, 2 mL water and 6 mL toluene, followed by a solution of230 mg (1.05 mmol) of di-tert-butyl dicarbonate. The course of thereaction was followed by TLC (silica, 5% methanol in dichloromethane).Every 10 minutes an additional amount of the dicarbonate was added untilall starting material had reacted. A total of approximately 1.5equivalents were used. A mixture of water and dichloromethane (60 mLeach) was added, and, after thorough shaking, the layers were separated.The aqueous layer was extracted with an additional 30 mL ofdichloromethane. The combined organic extracts were dried with anhydrousmagnesium sulfate. During this procedure, the color of the solutionturned from pink to light yellow. Evaporation in vacuo gave 295 mg (83%)of tert-butyl-4-(6-acetyl-2-naphthyl)-1-piperazinecarboxylate, which, onrecrystallization from dichloromethane-petroleum ether mixture, meltedat 153-154° C. Anal. Calculated for C₂₁H₂₆N₂O₃: C, 71.16; H, 7.39; N,7.90. Found: C, 71.27; H, 7.60; N, 7.86. ¹H NMR: δ1.50 (s, 9H,—C(CH₃)₃), 2.68 (s, 3H, CH₃), 3.33 and 3.64 (t, J=4.9 Hz, 8H,piperazine), 7.10 (d, 1H, 5-H), 7.31 (dd, 1H, 7-H), 7.70 (d, 1H, 4-H),7.85 (d, 1H, 8-H), 7.97 (d, 1H, 3-H), 8.35 (d, 1H, 1-H); J_(5,7)=2 Hz,J_(7,8)=9 Hz, J_(3,4)=8.7 Hz.

Example 1(e) Preparation of2-[1-(6-piperazino-2-naphthyl)ethylidene]malononitrile

[0077]

[0078] tert-Butyl 4-(6-acetyl-2-naphthyl)-1-piperazinecarboxylate (177mg, 0.5 mmol), prepared as described in Example 1(d), was heated with 40mg (0.6 mmol) of malononitrile in 4 mL pyridine at 105-110° C. After 5.5hours, an additional 24 mg of malononitrile was added, and heating wascontinued for a total of 12 hours and 40 minutes. The mixture was cooledand evaporated in vacuo. Polar components of the mixture were removed bycolumn chromatography (70-230 mesh silica, φ20×120 mm, chloroform), andthe product,tert-butyl-4-[6-(2,2-dicyano-1-methylvinyl)-2-naphthy]-1-piperazinecarboxylate,was finally purified by radial chromatography (silica, 2 mm,chloroform). 155 mg (77%) oftert-butyl-4-[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl]-1-piperazinecarboxylatewere obtained, which, after recrystallization fromdichloromethane-petroleum ether mixture, melted at 169-171° C. Anal.Calculated for C₂₄H₂₆N₄O₂: C, 71.62; H, 6.51; N, 13.92. Found: C, 71.62;H, 6.66; N, 13.87. ¹H NMR: δ1.50 (s, 9H, —C(CH₃)₃), 2.72 (s, 3H, CH₃),3.34 and 3.34 (t, J=5.1 Hz, 8H, piperazine), 7.09 (d, 1H, 5-H), 7.33(dd, 1H 7-H), 7.58 (dd, 1H, 3-H), 7.74(d, 1H, 4-H), 7.81 (d, 1H, 8-H),8.02 (d, 1H, 1-H); J_(5,7)=2 Hz, J_(7,8)=9.1 Hz, J_(1,3)=2 Hz,J_(3,4)=9.1 Hz.

[0079] Whentert-butyl-4-[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl]-1-piperazinecarboxylatewas treated with TFA (trifluoroacetic acid) at room temperature, TLCshowed that the reaction was over in 5 minutes and gave a singleproduct, 2-[1-(6-piperazino-2-naphthyl)ethylidene]malononitrile. The TFAwas removed in vacuo at room temperature. ¹H NMR: δ2.72 (s, 3H, CH₃),3.50 and 3.63 (broad, 8H, piperazine), 7.18 (broad s, 1H, 5-H), 7.29 (d,1H, 7-H), 7.59 (d, 1H, 3-H), 7.79 (d, 1H, 4-H), 7.87 (d, 1H, 8-H), 8.04(s, 1H, 1-H), 9.0 (broad, 1.5H, NH and acid); J_(7,8)=8.8 Hz,J_(3,4)=8.4 Hz. ¹⁹F NMR: δ−76.2 (CF₃COO).

[0080] NMR of the residue revealed that the tert-butyloxycarbonyl grouphas been removed and that there was some TFA left (¹⁹F NMR).Dichloromethane (10 mL) was added and the solution was washed withsaturated NaHCO₃ solution, dried, and evaporated in vacuo. A lightyellow oil was obtained, which, on standing at room temperature, turneddark red. TLC showed that the change in color is due to decomposition of2-[1-(6-piperazino-2-naphthyl)ethylidene]malononitrile into severalproducts, the most intense spot being low-R_(f) red-orange. Selected ¹HNMR signals after neutralization: δ2.72 (s, 3H, CH₃), 3.09 and 3.35 (t,J=5 Hz, 8H, piperazine), 7.08 (s, 1H, 5-H), 8.02 (s, 1H, 1-H).

Example 1(f) Preparation of2-(1.1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene([F-18]FDDNP)

[0081]

[0082] A mixture of 4.15 g (55.5 mmol) NaHSO₃, 8 mL of water, 0.78 g(4.19 mmol) of 1-(6-hydroxy-2-naphthyl)-1-ethanone (prepared asdescribed in Example 1(b)), and 8 mL of 2-methylaminoethanol was heatedand stirred in a steel bomb at 140° C. for 28 hours. After cooling, themixture was distributed between ethyl acetate and water (500 mL and 200mL, respectively). The organic layer was dried and evaporated to leaveraw 1-(6-(2-hydroxyethyl-methylamino)-2-naphthyl)-1-ethanone (0.749 g,73%) of which was further purified by radial chromatography (4 mm SiO₂,CH₂Cl₂).

[0083] To a solution of 201 mg (0.83 mmol) of1-(6-2-hydroxyethyl-methylamino-2-naphthyl)-1-ethanone in pyridine (6mL), malononitrile (236 mg, 3.6 mmol) was added and the mixture washeated at 95° C. for 24 hours. The solvent was removed in vacuo, and theresidue was chromatographed by radial chromatography (4 mm SiO₂ 1%MeOH/CH₂Cl₂) to give 150 mg (73%) of2-(1,1-dicyanopropen-2-yl)-6-2-hydroxyethyl)-methyl-amino)-naphthalene.

[0084] To the solution of2-(1,1-dicyanopropen-2-yl)-6-(2-hydroxyethyl)-methylamino)-naphthalene(120 mg, 0.41 mmol) in pyridine (5 mL), p-toluensulfonic anhydride wasadded (441 mg, 1.35 mmol). After stirring at room temperature for 1hour, pyridine was removed under vacuum, and the residue waschromatographed by radial chromatography (2 mm SiO₂, CH₂Cl₂) to give 183mg (80%) of2-(1,1-dicyanopropen-2-yl)-6-(2-tosyloxyethyl)-methylamino-naphthalene.

[0085] Radioactive ¹⁸F-fluoride 528.5 mCi from the cyclotron wastransferred into a solution of 19 mg of Kryptofix 2.2.2 and 0.75 mgpotassium carbonate in 50 μL of water and 300 μL of acetonitrile. Waterwas removed in a stream of nitrogen at 115° C. followed bycodistillation with acetonitrile (3×200 μL). The tosylate(2-(1,1-dicyanopropen-2-yl)-6-(2-tosyloxyethyl)-methylamino)-napthalene,4 mg) in 1 mL of acetonitrile was added, and the mixture was heated at85-86° C. for 20 minutes. After cooling, 1 mL of water was added, andthe mixture was transferred onto a C-18 Sep-Pak Cartridge, washed withdistilled water (3×4 mL) and eluted with CH₂Cl₂ (2×2.5 mL). Eluate wasdried by passing through a column packed with sodium sulfate and loadedonto a HPLC column (Whatman Partisil Silica 10, 500×10 mm, mL/minCH₂Cl₂:hexane=7:3, UV detector @ 254 nm, radioactivity detector). Eluatewas collected, appropriate fractions were combined, evaporated undervacuo to yield 50.7 m Ci (17%, corrected for decay) of the titledproduct which was formulated for injection. The synthesis was completein 50 minutes.

Example 2

[0086] Detection and labeling of β-amyloid plaques in vitro and in vivo,using brain tissue sections and rat brains, were conducted using thefollowing procedures.

[0087] A 2.1 mg/mL DDNP stock solution was prepared, which was adjustedto 8 mM in 100% ethanol. A DDNP working solution was prepared bydiluting the stock solution with distilled water in a ratio of1:100-1000 (stock solution:distilled water).

[0088] β-amyloid 250 μM (1.25 mg/mL in distilled water) was aggregatedat 37° C. for 48 hours. 5 μL were smeared on slides, air-dried and thenrehydrated with distilled water. Alternatively, Aβ-positive brain tissuesections were rehydrated with distilled water. DDNP working solution wasapplied to each slide for 30 minutes at room temperature. The slideswere washed three times for five minutes with distilled water. Theslides were coverslipped with fluorescent protectant mounding media(Vectashield™, available Vector Labs., Burlingame, Calif.) and observedunder a fluorescence microscope with a thioflavin S or FITC filter.

[0089] β-amyloid 250 μM (1.25 mg/mL in distilled water) was aggregatedat 37° C. for 48 hours to produce fibrils confirmed by smears. Threerats were anesthetized. 3 μL of a solution of Aβ fibrils (1.25 μg/μL)were injected unilaterally into the cortex of each rat (Bregma 0, AP-4.1mm, ML+2.0 mm, DV-3.1 mm). Then 3 μL of phosphate buffered saline (PBS)were injected into the contralateral side of each rat brain as a vehiclecontrol. After injection, the needle remained for 5 minutes to preventreflux, and then the cranial hole was sealed with bone wax. Eight daysafter β-amyloid injection into the rat brains, the rats were injectedwith 10 microliters of DDNP working solution (320 micromolar) preparedby diluting DDNP stock solution into 1.5% BSA (bovine serum albumin) inphosphate buffered saline, pH 7.2).

[0090] After one hour, the rats were cardiac perfused with PLP fixative(4% paraformaldehyde, 1% lysin in 0.05 M phosphate buffer, pH 7.4).Additional immersion fixation of rat brain was at 4° C. overnight withPLP fixative. The rat brains were washed with PBS, saturated in 10 and20% sucrose, and snap frozen in chilled isopentane (−70° C.) with liquidnitrogen. The brains were cryostat sectioned at 10 μM around theneedle-track and directly coverslipped with glycerol and fluorescenceprotectant (Vectashield™). The brain sections were observed with afluorescence microscope.

[0091]FIGS. 1A to 1F depict amyloid plaques labeled in sections frombrain of an AD patient and a transgenic mouse, demonstrating that DDNPis able to label amyloid plaques. FIGS. 2A to 2E depict labeledβ-amyloid plaques, demonstrating that DDNP passes the blood brainbarrier in rats.

[0092] It was found that DDNP readily labeled amyloid deposits incryostat and paraffin sections of AD brain tissue with a level ofsensitivity similar to thioflavin S. Use of DDNP has several advantagesover thioflavin S. Namely, the use of DDNP requires no pretreatmentsand, unlike thioflavin S, works with minimal washing and withoutformalin or paraformaldehyde fixation or differentiation of tissue.Stock solution can be kept in the freezer for six months and stillproduce acceptable results at 1/100 to 1/1,000 dilutions, eliminatingthe need to make the stock up fresh, as is required for thioflavin Slabeling.

Example 3

[0093] Labeling of human β-amyloid plaques and neurofibrillary tanglesin vivo were conducted using the following procedures.

[0094] A patient was placed in a tomograph to obtain brain dynamic PETimages. 8.0 mCi of2-(1.1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene([F18]FDDNP) (specific activity: 5-12 Ci/micromol; mass:˜1 nanomol),prepared as described in Example 1(f), were injected intravenously intothe arm of the patient. Dynamic acquisition data of brain images wererecorded simultaneously in forty-seven brain planes for two hours.

[0095] It was found that [F-18]FDDNP readily crosses the brain bloodbarrier and labels brain by structures in a manner consistent with thepresence of beta amyloid plaques and neurofibrillary tangles. Thepatient had previously had ¹⁸F-fluorodeoxyglucose (FDG)/positronemission tomography (PET) scans, as well as MRI scans to monitor brainatrophy. In areas where the maximum atrophy was observed in the MRIscans (low temporal and parietal lobes), maximum accumulation ofthe[F-18]FDDNP label was observed. In those areas, low glucosemetabolism (as measured with FDG/PET scans) was also observed.

Example 4

[0096] Labeling and detection of human β-amyloid plaques andneurofibrillary tangles in vivo were conducted using the followingprocedures. Ten human subjects, seven Alzheimer's diseased patients(ages 71 to 80) and three control patients (ages 62 to 82) were studied.The patients were positioned supine in an EXACT HR+962 tomograph(Siemens-CTI, Knoxville, Tenn.) with the imaging plane parallel to theorbito-meatal line. Venous catheterization was performed, and then[F-18]FDDNP (5-10 mCi) in human serum albumin (25%) was administered asa bolus via the venous catheter. Sequential emission scans were obtainedbeginning immediately after [F-18]FDDNP administration using thefollowing scan sequence: six 30 second scans, four 3 minute scans, five10 minute scans, and three 20 minute scans. Rapid venous blood samplingwas performed via the indwelling catheter in two subjects for inputfunction determination and plasma metabolite analysis.

[0097]FIG. 3A provides a PET-[F-18]FDDNP(2-(1.1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene)image of a brain cross-section through thehippocampus-amygdala-entorhinal/temporal cortex region of an Alzheimer'sdisease patient. The image was reconstructed from scanning data obtained30 to 60 minutes post [F-18]FDDNP injection. Co-registered PET-FDG (FDGis 2-[F-18]fluoro-2-deoxy-D-glucose) and MRI (proton relaxation times)images of the patient are also shown, in FIGS. 3B and 3C, respectively,to provide information about glucose metabolism and anatomicalstructures on the cross-section, respectively. The medial temporalregion appears darker in the [F-18]FDDNP scan (slower clearance) andlighter in the FDG scan (reduced glucose metabolism).

[0098]FIG. 4 demonstrates that estimated residence times of [F-18]FDDNPin Alzheimer's diseased patients are seen to be different from values incontrol patients. The residence time shown is in reference to that inthe pons, an area known to have limited involvement in Alzheimer'sdisease pathology. The residence time was calculated from the clearancerates of the tracer in affected regions of interest (ROI) and in thepons as:

Residence time=[1/clearance rate for affected ROI]−[1/clearance rate forpons]

[0099] Separate ROIs were defined in entorhinal cortex, hippocampus,lateral temporal cortex and pons. The region with the slowest clearancerate was used as the affected ROI in the calculation of the residencetime shown in FIG. 4.

[0100] It was found that after intravenous injection, [F-18]FDDNPcrosses the blood brain barrier readily in proportion to blood flow.Accumulation of radioactivity was followed by the differential regionalclearance of [F-18]FDDNP. A slower clearance was observed in brain areasreliably known to accumulate β-amyloid plaques and neurofibrillarytangles, specifically the hippocampus-amygdala-entorhinal complex, aswell as temporal and parietal cortex in more advanced states of thedisease. rCMRG1 measured with PET in these subjects were also consistentwith the expected β-amyloid plaque load and the possible presence ofneurofibrillary tangles. In these patients, brain areas with low glucosemetabolism were in general matched with high retention of [F-18]FDDNP.The hippocampus-amygdala-entorhinal cortex presented high retention ofactivity ([F-18]FDDNP) in most cases, even in patients with low severityof symptoms. A normal 82 year old volunteer presented deposition ofactivity in the hippocampus-amygdala-entorhinal complex in a PET studywith [F-18]FDDNP, and low rCMRG1 in the same areas, as measured withFDG, as shown in FIG. 4. These results are consistent with observationsthat elderly individuals without apparent signs of dementia may presentneurofibrillary pathology in the second layer of neurons of theentorhinal cortex and plaques in the hippocampal formation. Increasedseverity of symptoms was always accompanied with increased retention ofactivity, and slow clearance from temporal, parietal or frontal cortex,in agreement with expected Aβ and neurofibrillary tangles deposition inthese areas.

[0101] In vitro autoradiography using [F-18]FDDNP with brain specimensof Alzheimer diseased patients also demonstrated a distribution ofactivity consistent with the presence of β-amyloid plaques andneurofibrillary tangles. Binding was observed in hippocampus, temporaland parietal cortex matching results with immunostaining Aβ and tauantibodies. Since DDNP and its derivatives are fluorescent, anevaluation of the ability of [F-18]FDDNP to label β-amyloid plaques andneurofibrillary tangles in vitro was also performed with the same brainspecimens. In all Alzheimer's disease brain specimens, excellentvisualization of neurofibrillary tangles, amyloid peptides, and diffuseamyloid was produced with both DDNP and [F-18]FDDNP, matching resultswith thioflavin S (24) obtained with the same samples.

[0102] In FIG. 5, the central image was obtained by immunostaining aforty five micrometer cryostate temporal cortex section of anAlzheimer's disease patient incubated with AT8 (anti-phosphotau) and10G4 (anti-AB1-15) at 1:800. Insets are adjacent sections of the sameAlzheimer's disease brain specimen stained with FDDNP. Images weregenerated using fluorescent scanning microscopy. Green arrows indicateapproximate origin of inset with reference to central immunostainingsection. Beginning in upper left corner and moving clockwise, the insetsshow (1) neuritic plaques, (2) diffuse plaque, (3) vascular amyloid, (4)dense plaques and tangles, and (5) dense tangles.

[0103] This invention in its broader aspect is not limited to thespecific details shown and described herein. Departures from suchdetails may be made without departing from the principles of theinvention and without sacrificing its chief advantages.

We claim:
 1. A method for labeling structures selected from the groupconsisting of β-amyloid plaques and neurofibrillary tangles either invivo or in vitro, comprising contacting brain tissue, with a compound offormula (I):

wherein: R₁ is selected from the group consisting of —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C═C(CN)₂-alkyl,—C═C(CN)₂-alkylenyl-R₄

 wherein R₄ is a radical selected from the group consisting of alkyl,substituted alkyl, aryl and substituted aryl; R₅ is a radical selectedfrom the group consisting of —NH₂, —OH, —SH, —NH-alkyl, —NHR₄,—NH-alkylenyl-R₄, —O-alkyl, —O-alkylenyl-R₄, —S-alkyl, and—S-alkylenyl-R₄; R₆ is a radical selected from the group consisting of—CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)-halogen, —C(O)NH₂, —C(O)NH-alkyl,—C(O)NH-alkylenyl-R₄; R₇ is a radical selected from the group consistingof O, NH, and S; and R₈ is N, O or S; and R₂ and R₃ are eachindependently selected from the group consisting of alkyl andalkylenyl-R₁₀, wherein R₁₀ is selected from the group consisting of —OH,—OTs, halogen, spiperone, spiperone ketal, and spiperone-3-yl, or R₂ andR₃ together form a heterocyclic ring, optionally substituted with atleast one radical selected from the group consisting of alkyl, alkoxy,OH, OTs, halogen, alkyl-R₅, carbonyl, spiperone, spiperone ketal andspiperone-3-yl, and further wherein one or more of the hydrogen, halogenor carbon atoms are optionally replaced with a radiolabel.
 2. A methodaccording to claim 1, wherein the compound of formula (I) isradiolabeled with ¹⁸F or ¹²³I.
 3. A method for labeling structuresselected from the group consisting of β-amyloid plaques andneurofibrillary tangles either in vivo or in vitro, comprisingcontacting brain tissue, with a compound of formula (II):

wherein R₂ and R₃ are each independently selected from the groupconsisting of alkyl and alkylenyl-R₁₀, wherein R₁₀ is selected from thegroup consisting of —OH, —OTs, halogen, spiperone, spiperone ketal andspiperone-3-yl, or R₂ and R₃ together form a heterocyclic ring,optionally substituted with at least one radical selected from the groupconsisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R₁₀, carbonyl,spiperone, spiperone ketal and spiperone-3-yl, and R₉ is an alkyl, aryland substituted aryl; and further wherein one or more of the hydrogen,halogen or carbon atoms are optionally replaced with a radiolabel.
 4. Amethod according to claim 3, wherein the compound of formula (II) isradiolabeled with ¹⁸F or ¹²³I.
 5. A method according to claim 1, whereinthe compound of formula (I) is2-(1,1-dicyanopropen-2-yl)-6-dimethylaminonaphthalene, optionallycontaining a radiolabel.
 6. A method according to claim 5, wherein the2-(1,1-dicyanopropen-2-yl)-6-dimethylaminonaphthalene is radiolabeledwith ¹⁸F or ¹²³I.
 7. A method according to claim 1, wherein the compoundof formula (I) is2-(1,1-dicyanopropen-2-yl)-6-(2-ethyl)-methylamino)-naphthalene,optionally containing a radiolabel.
 8. A method according to claim 1,wherein the compound of formula (I) is2-(1,1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene.9. A method according to claim 1, further comprising determining whetherthe structures are labeled by observing the brain tissue with afluorescence microscope.
 10. A method according to claim 1, wherein thecompound according to claim 1 is radiolabeled.
 11. A method according toclaim 10, further comprising determining whether the structures arelabeled by observing the brain tissue by a method capable of detectingand depicting the distribution of the radiolabeled compound within thebody.
 12. A method according to claim 11, wherein the brain tissue isobserved using positron emission tomography.
 13. A method for diagnosingAlzheimer's disease in a patient, comprising: injecting into braintissue of the patient a compound of formula (I):

wherein: R₁ is selected from the group consisting of —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C═C(CN)₂-alkyl,—C═C(CN)₂-alkylenyl-R₄,

 wherein R₄ is a radical selected from the group consisting of alkyl,substituted alkyl, aryl and substituted aryl; R₅ is a radical selectedfrom the group consisting of —NH₂, —OH, —SH, —NH-alkyl, —NHR₄,—NH-alkylenyl-R₄, —O-alkyl, —O-alkylenyl-R₄, —S-alkyl, and—S-alkylenyl-R₄; R₆ is a radical selected from the group consisting of—CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)-halogen, —C(O)NH₂, —C(O)NH-alkyl,—C(O)NH-alkylenyl-R₄; R₇ is a radical selected from the group consistingof O, NH, and S; and R₈ is N, O or S; and R₂ and R₃ are eachindependently selected from the group consisting of alkyl andalkylenyl-R₁₀, wherein R₁₀ is selected from the group consisting of —OH,—OTs, halogen, spiperone, spiperone ketal, and spiperone-3-yl, or R₂ andR₃ together form a heterocyclic ring, optionally substituted with atleast one radical selected from the group consisting of alkyl, alkoxy,OH, OTs, halogen, alkyl-R₁₀, carbonyl, spiperone, spiperone ketal andspiperone-3-yl, and further wherein one or more of the hydrogen, halogenor carbon atoms are optionally replaced with a radiolabel; anddetermining whether the compound of formula (I) labeled structuresselected from the group consisting of β-amyloid plaques andneurofibrillary tangles.
 14. A method according to claim 13, wherein thecompound of formula (I) is radiolabeled with ¹⁸F or ¹²³I.
 15. A methodaccording to claim 13, wherein the compound of formula (I) is a compoundof formula (II):

wherein R₂ and R₃ are each independently selected from the groupconsisting of alkyl and alkylenyl-R₁₀, wherein R₁₀ is selected from thegroup consisting of —OH, —OTs, halogen, spiperone, spiperone ketal andspiperone-3-yl, or R₂ and R₃ together form a heterocyclic ring,optionally substituted with at least one radical selected from the groupconsisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R₁₀, carbonyl,spiperone, spiperone ketal and spiperone-3-yl, and R₉ is an alkyl, aryland substituted aryl.
 16. A method according to claim 15, wherein thecompound of formula (II) is radiolabeled with ¹⁸F or ¹²³I.
 17. A methodaccording to claim 13, wherein the compound of formula (I) is(2-(1.1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene).18. A method according to claim 13, wherein the determining stepcomprises observing the brain tissue by a method capable of detectingand depicting the distribution of the radiolabeled compound within thebody.
 19. A method according to claim 13, wherein the brain tissue isobserved using positron emission tomography (PET).
 20. A methodaccording to claim 13, wherein the brain tissue is observed using singlephoton emission computed tomography (SPECT).
 21. A method for labelingβ-amyloid plaques, either in vivo or in vitro, comprising contactingbrain tissue, with a compound of formula (I):

wherein: R₁ is selected from the group consisting of —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C═C(CN)₂-alkyl,—C═C(CN)₂-alkylenyl-R₄,

 wherein R₄ is a radical selected from the group consisting of alkyl,substituted, alkyl, aryl and substituted aryl; R₅ is a radical selectedfrom the group consisting of —NH₂, —OH, —SH, —NH-alkyl, —NHR₄,—NH-alkylenyl-R₄, —O-alkyl, —O-alkylenyl-R₄, —S-alkyl, and—S-alkylenyl-R₄; R₆ is a radical selected from the group consisting of—CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)-halogen, —C(O)NH₂, —C(O)NH-alkyl,—C(O)NH-alkylenyl-R₄; R₇ is a radical selected from the group consistingof O, NH, and S; and R₈ is N, O or S; and R₂ and R₃ are eachindependently selected from the group consisting of alkyl andalkylenyl-R₁₀, wherein R₁₀ is selected from the group consisting of —OH,—OTs, halogen, spiperone, spiperone ketal, and spiperone-3-yl, or R₂ andR₃ together form a heterocyclic ring, optionally substituted with atleast one radical selected from the group consisting of alkyl, alkoxy,OH, OTs, halogen, alkyl-R₁₀, carbonyl, spiperone, spiperone ketal andspiperone-3-yl, and further wherein one or more of the hydrogen, halogenor carbon atoms are optionally replaced with a radiolabel.
 22. Acomposition comprising a compound of formula (I):

wherein: R₁ is selected from the group consisting of —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C═C(CN)₂-alkyl,—C═C(CN)₂-alkylenyl-R₄

 wherein R₄ is a radical selected from the group consisting of alkyl,substituted alkyl, aryl and substituted aryl; R₅ is a radical selectedfrom the group consisting of —NH₂, —OH, —SH, —NH-alkyl, —NHR₄,—NH-alkylenyl-R₄, —O-alkyl, —O-alkylenyl-R₄, —S-alkyl, and—S-alkylenyl-R₄; R₆ is a radical selected from the group consisting of—CN, —COOH, —C(O)O-alkyl, —C(O)O-alkylenyl-R₄, —C(O)-alkyl,—C(O)-alkylenyl-R₄, —C(O)-halogen, —C(O)NH₂, —C(O)NH-alkyl,—C(O)NH-alkylenyl-R₄; R₇ is a radical selected from the group consistingof O, NH, and S; and R₈ is N, O or S; and R₂ is selected from the groupconsisting of alkyl and alkylenyl-R₁₀ and R₃ is alkylenyl-R₁₀, whereinR₁₀ is selected from the group consisting of —OH, —OTs, halogen,spiperone, spiperone ketal, and spiperone-3-yl, or R₂ and R₃ togetherform a heterocyclic ring, optionally substituted with at least oneradical selected from the group consisting of alkyl, alkoxy, OH, OTs,halogen, alkyl-R₁₀, carbonyl, spiperone, spiperone ketal andspiperone-3-yl, and further wherein one or more of the hydrogen, halogenor carbon atoms are optionally replaced with a radiolabel.
 23. Acomposition according to claim 22, wherein the compound of formula (I)is radiolabeled with ¹⁸F or ¹²³I.
 24. A composition according to claim22, comprising a compound of formula (II):

wherein R₂ is selected from the group consisting of alkyl andalkylenyl-R₁₀ and R₁₀ is alkylenyl-R₁₀, wherein R₁₀ is selected from thegroup consisting of —OH, —OTs, halogen, spiperone, spiperone ketal andspiperone-3-yl, or R₂ and R₃ together form a heterocyclic ring,optionally substituted with at least one radical selected from the groupconsisting of alkyl, alkoxy, OH, OTs, halogen, alkylenyl-R₁₀, carbonyl,spiperone, spiperone ketal and spiperone-3-yl, and R₉ is an alkyl group;or a pharmaceutically acceptable salt or solvate thereof; and furtherwherein one or more of the hydrogen, halogen or carbon atoms areoptionally replaced with a radiolabel.
 25. A composition according toclaim 24, wherein the compound of formula (II) is radiolabeled with ¹⁸For ¹²³I.
 26. A composition according to claim 22, wherein the compoundof formula (I) is2-(1,1-dicyanopropen-2-yl)-6-(2-[¹⁸F]-fluoroethyl)-methylamino)-naphthalene.